Bone morphogenetic proteins (BMPs) belong to the TGF-βsuperfamily of the secreted growth and differentiation factors. The BMP subfamily of the TGF-β superfamily includes at least fifteen proteins, including BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14 and BMP-15.
It has been reported that BMPs regulate the growth and differentiation of cells such as osteogenic cells, and particularly BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 and BMP-8 are growth and differentiation factors that can induce ectopic bone formation alone in vitro and in vivo. Also, BMP-2 can direct the development of neural crest cells into neural phenotypes, and BMP-4 and BMP-7 can induce a sympathetic adrenergic phenotype. It has been reported that a heterodimer composed of BMP-4 and BMP-7 is a potent inducer of mesoderm (see A. Suzuki, E. Kaneko, J. Maeda and N. Ueno, “Mesoderm induction by BMP-4 and -7 heterodimers,” Biochem. Biophys. Res. Commun., vol. 232, pp. 153-156, March 1997).
In addition, BMPs can form either homodimers composed of monomers of a single BMP subfamily member or heterodimers composed of monomers of two different BMP subfamily members linked by a disulfide bond in a head-to-tail arrangement (see K. Azari, B. A. Doll, C. Sfeir, Y. Mu and J. O. Hollinger, “Therapeutic potential of bone morphogenetic proteins,” Expert Opinion on Investigational Drugs, vol. 10, pp. 1677-1686, September 2001). Also, it was reported that BMP dimers such as BMP-2 homodimers and BMP-2/7 heterodimers are active in vivo (see D. I. Israel, J. Nove, K. M. Kerns, I. K. Moutsatsos and R. J. Kaufman, “Expression and Characterization of Bone Morphogenetic Protein-2 in Chinese Hamster Ovary Cells,” Growth Factors, vol. 7, pp. 139-150, 1992; M. F. Mehler, P. C. Mabie, D. M. Zhang and J. A. Kessler, “Bone morphogenetic proteins in the nervous system,” Trends Neurosci., vol. 20, pp. 309-317, 1997; T. K. Sampath, J. E. Coughlin and R. M. Whetstone, “Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-b superfamily,” J. Biol. Chem., vol. 265, pp. 13198-13205, August 1990; D. I. Israel; J. Nove, K. M. Kerns, R. J. Kaufman, V. Rosen, K. A. Cox and J. M. Wozney, “Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo,” Growth Factors, vol. 13, pp. 291-300, 1996).
Particularly, it is known in the art that BMP-2 belonging to the TGF-β superfamily of proteins plays an important role in the development of bone and cartilage, and potentially induce osteoblastic differentiation of a variety of cell types (see “Regulation of human cranial osteoblast phenotype by FGF-2, FGFR-2 and BMP2 signaling”, Marie P J, Debiais F, Hay E, 2002). Also, BMP-2 acts to stimulate bone production, and products that induce new bone formation by implanting BMP-2 into collagen sponges are being marketed.
In connection with BMP-2 playing an important role in bone tissue regeneration as described above, various studies have been conducted. Accordingly, it is required to remove proteins or impurities other than BMP-2 from cell culture fluids containing BMP-2 and to obtain BMP-2 from the cell culture fluids in high purity and high yield.
In the prior art, in order to purify BMP-2 from culture fluids of cell, etc. in high purity and obtain it in high yield, several steps of chromatography were combined and performed depending on charges, ligands, the degree of hydrophobicity or size. The type of chromatography used in purification is very diverse, and the purity and yield of BMP-2 greatly vary depending on the type of chromatography. However, as the time for purification of BMP-2 increases and the number of steps for the purification increases, there is a problem in that BMP-2 to be obtained as a final product is degraded by any protease during purification steps or lost during the purification process, resulting in a decrease in the final M yield of BMP-2. Moreover, even if anyone tries to reduce the time and the number of steps for purifying BPM-2 in order to solve this problem, the purity of the finally recovered BMP-2 should not be low.